In the life cycle of HIV, the Rev protein regulates the temporal switch from the early regulatory to the late lytic phase. The Rev regulatory protein of HIV is a basic nuclear protein that concentrates in the nucleoli and activates the viral RNAs, by binding to a highly structured RRE (Rev Responsive Element) RNA. Our studies of Rev and RRE RNA were targeted i) to explore the roles of RRE other than Rev binding in the activation process, ii) to analyze the various functional motifs of the Rev protein; and iii) to identify and characterize the function of the putative cellular factors that may bind RRE RNA, Rev, or both. We have completed an exhaustive analysis of mutants in the various functional motifs of Rev both in the context of Rev and of Rev/MS2 phage coat protein chimera. The latter chimera allowed us to dissociate RNA binding from activation, and thus evaluate the roles of RRE RNA in the biochemical aspects of Rev trans-activation. From these genetic studies, we propose that poly-arginine insertion near the N-terminus of Rev promotes nucleolar targeting in a context sensitive manner, and the Rev sequence immediately flanking the 9 arginines (residues 24-35, and residues 36-50) is required for RRE RNA binding. We have further characterized the biochemical properties of HIV-1 Rev Responsive Element (RRE) RNA binding cellular factor (RBF) that was identical to the HIV TAR RNA bindingprotein (TRBP). TRBP bore significant sequence homology with many cellular and viral ds RNA binding proteins including the interferon inducible ds RNA activated protein kinase, PKR. TRBP was a competitive inhibitor of ds RNA activation of interferon induced PKR kinase in vitro and in vivo. PKR inhibition may be mediated by heterodimerization between PKR and TRBP and may require a small ds RNA. We have completed our analysis of the gene structure transcriptional map of TRBP and defined its sub- cellular location. We have extended our studies on the effect of HIV-1 NEF protein on T lymphocytre CD4 receptor. At low levels of Nef expression, the effect on cell surface expression was profound; increasing amounts of Nef resulted in a dose-dependent reduction in the steady-state levels of CD that approached 90%.